Chromosomal rearrangements are a type of genomic variation, which have been long been associated with genetic diseases such as Down syndrome (a trisomy), Jacobsen syndrome (a deletion) and Burkitt's lymphoma (a translocation) and have traditionally been studied via karyotype analysis. Genomic instability also leads to complex patterns of chromosomal rearrangements in certain cells, such as, for example, cancer cells.
Standard cytogenetic assays such as Giemsa (G) banding have identified numerous cancer-specific translocations and chromosomal abnormalities in cancer cells such as the Philadelphia (t9,22) chromosome. Improvements in cytogenetic banding and visualization such as M banding and spectral karyotyping (SKY) have enabled detailed analyses on a chromosome by chromosome basis of inversions and translocations, as well as the identification of regions of loss in cancers of interest. Fluorescence in situ hybridization (FISH) further allows for the detection of the presence or absence of specific DNA sequences on chromosomes by using fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of complementarity. All of these methods, however, have limited resolution since probes are generated from large pieces of DNA (flow-sorted chromosomes or bacterial artificial chromosomes for SKY and FISH, respectively). Because these probes are generated over very large regions of the genome, microtranslocations and microinversions cannot be resolved by current methods. The large templates from which probes are generated also present another disadvantage, in that both SKY and FISH probes contain repetitive DNA elements that are inherent in the large template DNA fragments. Thus, there has been an increasing need to understand more subtle chromosomal defects with substantially improved resolution, and without a priori knowledge of their location. A large unmet need exists to develop technical methods that detect novel, specific chromosomal abnormalities.
In one aspect, there is a need to avoid non-specific amplification of starting probes, which can lead to random amplification bias. There is also a need to create probes of a designated length consistently as fragments generated in current PCR processes are often too long to be used effectively in FISH, such that they require partial digestion by restriction enzymes which is difficult to control. There is also a need to target chromosomal regions by color on a very fine level such that microduplications, microinversions and microdeletions can be detected. Current techniques allow for painting of chromosomes in sections, however, the smallest unit that can be painted in one color is 10 megabases. There is also a need for utilization of standard laboratory equipment for the visual detection of signals from labeled probes such that special filters, software and processing steps are not required.
Certain aspects of this disclosure address these needs and describe methods and kits for practicing the same.